The active substances ursodesoxycholic acid (UDCA) and the corresponding diastereomer chenodesoxycholic acid (CDCA) have for many years been used for the medicinal treatment of gallstone problems. The two compounds differ only in the configuration of the hydroxy group on C atom 7 (UDCA: (3 configuration, CDCA: a configuration). For the production of UDCA, various methods are described in the state of the art, which are carried out purely chemically or consist of a combination of chemical and enzymatic process steps. In each case the starting point is cholic acid (CA) or CDCA produced from cholic acid.
The classical chemical method for UDCA production is shown schematically in FIG. 1a. A serious disadvantage of the classical method inter alia is as follows: because the chemical oxidation is not selective, the carboxy group and the 3α and 7α-hydroxy group must be protected by esterification.
An alternative chemical/enzymatic method based on the use of the enzyme 12α-hydroxy-steroid dehydrogenase (12α-HSDH) is shown schematically in FIG. 1b and is, for example, described in PCT/EP2009/002190 from the present applicant. In this alternative method, the 12α-HSDH selectively oxidizes CA to 12-keto-CDCA. The two protection steps necessary according to the classical chemical method are thereby rendered superfluous.
Further, Monti, D., et al., (One-Pot Multienzymatic Synthesis of 12-Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row. Advanced Synthesis & Catalysis, 2009) describe an alternative enzymatic-chemical method that is shown schematically in FIG. 1c. In this alternative, the CA is first oxidized by 7α-HSDH from Bacteroides fragilis ATCC 25285 (Zhu, D., et al., Enzymatic enantioselective reduction of α-ketoesters by a thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis. Tetrahedron, 2006. 62(18): p. 4535-4539) and 12α-HSDH to 7,12-diketo-LCA. These two enzymes are each NADH-dependent. After the reduction by 7β-HSDH (NADPH-dependent) from Clostridium absonum ATCC 27555 (DSM 599) (MacDonald, I. A. and P. D. Roach, Bile induction of 7 alpha-and 7 beta-hydroxysteroid dehydrogenases in Clostridium absonum. Biochim Biophys Acta, 1981. 665(2): p. 262-9), 12-keto-UDCA is formed. The final product is obtained by Wolff-Kishner reduction. Disadvantages in this method are that because of the equilibrium position of the catalyzed reaction a complete conversion is not possible, and that for the first step of the conversion two different enzymes must be used, which increases the process cost. For cofactor regeneration, lactate dehydrogenase (LDH; for regeneration of NAD+) and glucose dehydrogenase (GlcDH, for regeneration of NADPH) are used. A disadvantage in the cofactor regeneration used there is that the coproduct formed can only be removed from the reaction mixture with great difficulty, so that the reaction equilibrium cannot be favorably influenced, which results in incomplete conversion of the educt.
A 7β-HSDH from the strain Collinsella aerofaciens ATCC 25986 (DSM 3979; formerly Eubacterium aerofaciens) was described in 1982 by Hirano and Masuda (Hirano, S. and N. Masuda, Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens. Appl Environ Microbiol, 1982. 43(5): p. 1057-63). Sequence information on this enzyme was not disclosed. The molecular weight determined by gel filtration was 45,000 Da (see Hirano, page 1059, left-hand column). Further, for the enzyme there, the reduction of the 7-oxo group to the 7β-hydroxy group could not be observed (see Hirano, page 1061, discussion 1st paragraph). Similarly, Hirano et al also disclosed KM and Vmax values only for NADP+ (0.4 and 0.2 respectively) but not for NADPH. Those skilled in the art thus recognize that the enzyme described by Hirano et al is not suitable for the catalysis of the reduction of DHCA in the 7 position to 3,12-diketo-7β-CA.
Although the genome of Collinsella aerofaciens ATCC 25986 was already sequenced in 2007, none of the analyzed sequence motifs therein could be assigned to a potential short-chain dehydrogenase, to which enzyme family the HSDH enzymes belong.
In Biotechnology Letters 1992, 14,12, 1131-1135, Carrea et al describe a method for the production of UDCA from CA with the use of an enriched 7β-HSDH and a 3α-HSDH. However, a disadvantage in this method is the use of a 7β-HSDH from the pathogenic microorganism Clostridium absonum, and the need preparatively to purify the enzyme from the bacterial extract which additionally possesses undesired 7α-HSDH activity.
Hence the purpose of the invention is the provision of a novel method for the production of UDCA which avoids the aforesaid disadvantages. In particular, a novel enzyme which catalyzes the stereospecific reduction of DHCA in the 7-position to 3,12-diketo-7β-CA should be provided.
A further purpose consists in the provision of novel 7β-HSDH enzymes which for example are usable in the preparation of UDCA, and in particular catalyze the stereo- and enantioselective oxidation/reduction of cholic acid derivatives in the 7-position.